Proteolysis Targeting Chimeras (PROTACs) are event-driven bifunctional small-molecules that simultaneously engage an E3 ubiquitin ligase and a protein of interest (POI). Ternary complex formation between POI-PROTAC- E3 ligase, results in E3-ligase mediated POI ubiquitination and subsequent degradation of the POI by the 26S proteasome. The next innovation for PROTAC technology is the induction of tumor-specific protein degradation. PROTACs that induce degradation selectively in tumor cells would likely have improved therapeutic utility due to decreased off-target cytotoxicity. Currently, the E3 ligases most commonly hijacked for PROTAC-mediated POI degradation, von Hippel-Lindau, Cereblon, and Mouse double minute 2 homolog, are expressed in both cancerous and untransformed tissues. Therefore, new E3 ligase recruiting elements (E3REs) that engage E3 ligases with tumor-specific expression must be developed to impart tumor-specificity. Type I Melanoma Antigen Gene (MAGE) family proteins are cancer testis antigens, whose expression is restricted to the male germ line, but can be re-expressed in cancers. MAGE-A3 binds TRIM28, a ubiquitously expressed protein with E3 ligase activity, to form an oncogenic tumor-specific E3 ligase complex. A PROTAC harboring a MAGE-A3 E3RE may be able to recruit MAGE-A3/TRIM28 and induce protein degradation in a tumor-specific manner. MAGE proteins bind their cognate E3 ligases and substrates via a conserved MAGE homology domain (MHD). Using Schrdinger Glide docking software, we screened >60,000 compounds against the recently resolved structure of the MAGE-A3 MHD to identify ligands in silico that are predicted to disrupt MAGE-A3- substrate binding. We have identified a subset of lead-like compounds using intrinsic tryptophan fluorescence and are currently corroborating these findings via orthogonal biophysical assays such as isothermal calorimetry, and various NMR-based strategies. A structure-activity relationship study on bona-fide MAGE-A3 binders will then be performed to improve solubility, increase affinity, and identify (a) potential vector(s) for linker attachment in subsequent PROTAC development. Once tight-binding MAGE-A3 ligands have been developed, we will synthesize MAGE-A3-based-HaloPROTACs and test their ability to degrade HaloTag7-GFP in a MAGE-A3- dependent manner. Subsequently, we will further test the utility of recruiting MAGE-A3/TRIM28 E3 ligase complex by targeting Bromodomain-containing protein 4 (BRD4) for MAGE based-PROTAC mediated degradation. Induction of tumor-specific degradation of BRD4 and induction of apoptosis in a tumor-specific manner by our MAGE-A3 based-PROTACs will be evaluated. Overall, this project will determine the MAGE- A3/TRIM28 E3 ligase complex induce tumor-specific protein degradation. Additionally, development of a new E3RE will help spark excitement for identification of novel E3REs for other E3 ligases, thereby greatly expanding the number of E3 ligase amenable to the PROTAC technology. Moreover, PROTACs created during this project may serve as the starting point for the future development of a tumor-specific therapy.